Acyl coenzyme A:cholesterol acyltransferase (ACAT) utilizes two Iipophilic substrates, long-chain fatty acyl coenzyme A and cholesterol, to catalyze the formation of a neutral lipid cholesteryl ester (CE). At the single cell level, ACAT controls the cellular membrane cholesterol level by converting excess cholesterol into CEs. In cells involved in lipoprotein assembly, ACAT supplies CEs as part of the neutral lipid core in lipoproteins. Under pathophysiological conditions, ACAT is involved in forming CE-rich foam cells in the atherosclerotic plaques. The long-term goal of the PI of this application is to gain understanding of how this enzyme works at the molecular level. ACAT is a membrane bound enzyme located in the endoplasmic reticulum in minute quantity. The Pl's laboratory identified the first ACAT gene (human ACAT1) by functional complementation. The cloned human ACAT1 expressed in CHO cells and in insect cells has been solubilized by detergent and purified to homogeneity. At present, human ACAT1 is the only member of the membrane bound acyltransferase superfamily (comprised of at least 20 in numbers) that has been purified to homogeneity. The purified enzyme is shown to be under allosteric control by cholesterol. The enzyme is a homotetramer with multiple transmembrane domains. With various molecular reagents now available, in the current proposal, we request funds to test two hypotheses: A. Catalysis of ACAT1 may be completed within the plane of the ER membrane. B. ACAT1 may contain an allosteric sterol activator site in addition to a sterol substrate site. We enlist 3 Specific Aims: 1. To biochemically characterize the fatty acyI-CoA hydrolase activity intrinsically associated with hACAT1. 2. To probe the environment of the hydrophobic peptides (a.a. 446-468) comprising the putative ACAT active site. 3. To test the two sterol binding domain hypothesis.